Diagnostic Virology

• Diagnostic virology


  The study of diagnosing viral infections
• Specimen selection, collection & processing

  • Clinical & epidemiological consideration are important in the selection of specimen type & the determination of possible viral agents
  • Collect specimen from the source of disease as early as possible because the viral titer is highest during the first 4 days after the onset of symptoms except enteroviruses, adenovirus & Cytomegalovirus
  • It is best to sample the infected site directly
  • Viral transport medium is recommended for most specimens
  • Calcium alginate may bind & inactivate the virus & charcoal is toxic to several viruses thus should be avoided in the collection of viral specimen
  • Prompt transport enhance the detection of the virus but if a delay in processing the specimen is anticipated, specimen should be held at 4°C but not frozen
• Specimen that should be collected

  • Viral Infections of the respiratory tract
  • Suspected cases of Aseptic meningitis
  • Encephalitis
  • Skin Infections
  • Cytomegalovirus infections
  • Herpes simplex virus infections
  • Viral gastroenteritis
  • Ophthalmic viral infections
  • Congenital or neonatal viral pathogens
  • Viral agents associated with lymphadenopathy
• Laboratory methods in virology

  • Direct examination of clinical specimens
  • Virus isolation
  • Serological tests
• Direct examination of clinical specimens

  • Cytology
  • Electron microscopy
  • Immunodiagnosis
  • Nucleic acid probes & polymerase chain reaction (PCR) assays
• Virus isolation


  1. Using tissue culture cells

  2. Using embryonated eggs

  3. Using animal hosts
• Serological tests


  • Used primarily to detect immune status and to make the diagnosis of infections in situations in which the virus cannot be cultivated in cell culture or detected by immunoassay
  • An immune status test measures whether or not a patient has been infected by a particular virus in the past
  • A positive result with a sensitive virus-specific IgG test indicates past infection
  • Detection of virus-specific IgM in an acute phase specimen collected at least 10 to 14 days after the onset of infection indicates current or very recent infection
  • Detection of a fourfold antibody rise between acute and convalescent sera are also indicative of current or recent infection
  • Serologic methods routinely used to detect antiviral antibody: Complement fixation, Enzyme-linked immunoassay, Indirect immunofluorescence, Anticomplement immunofluorescence (ACIF)
• Difficulty of treatment


  • Close relationship & dependence of viruses upon host cell biosynthetic processes
  • Total virus multiplication in the host is nearly complete by the time symptoms appear
  • Emergence of drug resistant mutants
• Site of action of antiviral agents in viral multiplication

  1. Early events like entry & uncoating

  2. Nucleic acid synthesis by viral DNA & RNA polymerase

  3. Protein synthesis directed by viral mRNA

  4. Cleavage of precursor polypeptide

  5. Assembly of the virus

  6. Release of viral particle
• Prevention of viral infections

  • Chemoprophylaxis
  • Use of interferon
  • Immunization
• Active immunization


  • Live virus vaccine
  • Killed virus vaccine
  • Virion subunit vaccine
  • Viral polypeptides
• Passive immunization


  • Human immune serum
  • Hyperimmune gamma globulin
• Specimen selection, collection, and processing

  • Clinical and epidemiological consideration are important in the selection of specimen type and the determination of possible viral agents
  • Collect specimen from the source of disease as early as possible because the viral titer is highest during the first four (4) days after the onset of symptoms except enteroviruses, adenovirus, and cytomegalovirus
  • It is best to sample the infected site directly: vesicles or rash for skin infection, throat culture for respiratory tract infections, multiple sites for disseminated infections except for certain CNS infections where the virus is shed in stool or found in throat
  • Cotton swab with calcium alginate may bind and inactivate the virus and charcoal is toxic to several viruses thus should be avoided in the collection of viral specimen. Transport medium is necessary
  • Viral transport medium is recommended for most specimens. This medium contains buffered saline, a protein stabilizer, and antibiotics such as Penicillin, Vancomycin, Bacitracin, Streptomycin, and Amphoterecin B
  • Prompt transport enhance the detection of the virus but if a delay in processing the specimen is anticipated, specimen should be held at 4°C but not frozen
• Specimen that should be collected

  • Nasopharyngeal aspirates, secretion, or swabs
  • Sputum
  • Throat swabs
  • Bronchial washings
  • CSF
  • Throat swab
  • Stool culture
  • Brain biopsy
  • Blood
  • Vesicular fluid
  • Throat swabs or washings
  • Stool culture
  • Urine
  • EDTA-anticoagulated blood
  • Serum
  • Vesicular fluid
  • Endocervical swabs
  • Stools
  • Rectal swabs
  • Conjunctival swabs
  • Corneal or conjunctival scrapings
  • Urine
  • Throat swabs
  • Serum
  • Urine
  • Throat swabs
  • Serum
• Direct examination of clinical specimens

  • May be done on sections of tissue biopsies, tissue imprints or smears, blood, cerebrospinal fluid, urine, throat swabs, feces, and saliva
  • Should be performed only on those specimens likely to contain the virus
  • The virus itself can be seen or there are cells that will indicate the presence of the virus

  
• Cytology


  • Most readily available rapid technique for the detection of virus (presence of viral inclusion, cell lysis, cell morphology, and syncytia formation indicates presence of virus)
  • Can be done through Papanicolau (Pap) smear or Giemsa stained smears
  • Less sensitive than culture but is especially helpful for those viruses that are difficult or dangerous to isolate in the laboratory
• Cytological indicators of viral infection

  • Guarnieri bodies (Smallpox)
  • Negri bodies (Rabies)
  • Councilman bodies (Yellow fever)
  • Type B inclusion / Cowdry bodies (Cytomegalovirus)
  • Multinucleated giant cell

  
• Electron microscopy


  • Most helpful for the detection of viruses that do not grow readily in cell culture and works best if the titer of virus is at least 106 or 107 particles per milliliter
• Immune electron microscopy


  • Allows visualization of virus particles present in numbers too small for easy direct detection
• Immunodiagnosis techniques


  • Fluorescent antibody
  • Enzyme immunoassay
  • Radioimmunoassay
  • Latex agglutination
  • Immunoperoxidase test
• Fluorescent antibody test


  • Antibody is labeled with a fluorescent dye, and it reacts with an antigen so that what is seen is the immunofluorescence of the antigen-antibody reaction
  • Direct and indirect fluorescent antibody tests for antigen detection
• Fluorescent antibody staining of virus-infected cells

  • Influenza virus
  • Adenovirus
  • Varicella-zoster virus
  • Herpes simplex virus
  • Respiratory syncytial virus
  • Parainfluenza virus
  • Mumps virus
  • Measles virus
• Enzyme linked immunosorbent assay (ELISA)

  • Antibody is adsorbed to well
  • Patient sample is added; complementary antigen binds to antibody
  • Enzyme-linked antibody specific for test antigen is added and binds to antigen, forming a sandwich
  • Enzyme substrate is added, and reaction produces a product that causes a visible color change
• Radioimmunoassay


  Antigen or antibody is labeled to the radioactive material (made to react the bound antigen, which is made to react with an antibody)
• Latex agglutination


  • Antibody is bound to a latex bead and this is made to react with the antigen
  • The reaction is visualized as an agglutination and because of the latex bead
• Nucleic acid probes


  • Short segments of DNA that are designed to hybridize with complementary viral DNA or RNA segment
  • The probe is labeled with fluorescent, chromogenic or radioactive tag that allows detection if hybridization occurs
  • The most useful test in situations in which culture is slow or not possible and immunoassays lack sensitivity or specificity
• Polymerase chain reaction (PCR)


  • Method that duplicates short DNA segments thousand to a million fold
  • DNA fragments can be identified with a specific probe, but are too few in number in the original specimens to be detected, can be duplicated using PCR. This provides the probe with enough target to readily identify the presence of specific viruses
  • A duplicate of the DNA is made so that it can be visualized

  
• Tissue culture cells (primary cell line)

  • Routinely used for growing viruses
  • Primary cell culture: Normal cells freshly taken from body and cultured, limited growth (can be used for limited number of times)
  • Diploid cell strains: Cells of single type (fibroblast cells) that can be subcultivated for limited number of times, mostly 50
  • Continuous cell lines: Malignant cells, indefinite subcultivation
  • Tissue is treated with enzymes (culture medium with appropriate osmotic pressure, nutrients, and growth factors allows cell to grow)
  • Growth is observed: To observe degeneration and lysis of infected cells in the monolayer of cells, Areas where virus-infected cells have been destroyed show up as clear, well-defined patches in the cell sheet (plaque)
• Embryonated eggs


  • Chicken, duck, and turkey eggs are the most common choices for inoculation
  • Amniotic inoculation: inoculated into the amniotic sac
  • Chorioallantoic membrane inoculation: inoculated into the corioallantoic membrane
  • Yolk sac inoculation: inoculated into the yolk sac
• Growth medium - Minimum Essential Medium (MEM)


  Contains essential amino acids, vitamins, salts, glucose, and bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics, and phenol red indicator (as indicator)
• When cell culture bottles or tubes are examined in the laboratory

  • Normal cells can be seen
  • If there is a virus infected cell, inclusion body, fused cell (multinucleated giant cells), cell lysis, or transformed cell can be seen

  
• Growth is observed


  1. To observe degeneration and lysis of infected cells in the monolayer of cells

  2. Areas where virus-infected cells have been destroyed show up as clear, well-defined patches in the cell sheet (plaque)
• Inoculation to embryonated eggs results in

  • Death of the embryo
  • Defects in embryonic development
  • Pocks formation (Refers to localized areas of damage in the membrane (discrete opaque spots))
• If no changes occur in the embryo, use embryonic fluid and tissues
• Best time to inoculate animals

  • During the onset and acute phase of disease
• Serological test


  Used primarily to detect immune status and to make the diagnosis of infections in situations in which the virus cannot be cultivated in cell culture or detected by immunoassay
• Immune status test


  Measures whether or not a patient has been infected by a particular virus in the past
• Positive result with a sensitive virus-specific IgG test


  Indicates past infection
• Two approaches helpful in diagnosis of active disease

  1. Detection of virus-specific IgM in an acute phase specimen collected at least 10 to 14 days after the onset of infection (Indicates current or very recent infection)

  2. Detection of a fourfold antibody rise between acute and convalescent sera (Also indicative of current or recent infection)
• Rising titer of antibody in paired sample of sera is diagnostic